PROJECT SUMMARY/ABSTRACT The use of solid scaffolds that provide cells with mechanical and chemical cues is a promising approach for guiding tissue repair and promoting tissue-scaffold integration. It is becoming increasingly clear that tuning scaffold rigidity is a powerful way to control cell function, but how scaffold rigidity regulates gene expression is not well-understood. Previously, we tested the hypothesis that substrate rigidity controls gene expression by tuning nuclear tension. We took advantage of the fact that the LINC (linker of nucleoskeleton-to-cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substrate rigidity. Our results were the first to show that the LINC complex facilitates mechano-regulation of expression across the genome. Combined with myosin inhibition studies, we were able to identify genes that depend on nuclear tension for their mechanosensitivity. In this continuation, we propose to identify molecular mechanisms for these highly novel findings. Two specific aims are proposed: Aim 1: To identify the nuclear molecular linkers necessary for rigidity-mediated control of gene expression. Aim 2: To determine the mechanisms by which nuclear-cytoskeletal linkage regulates gene mechanosensitivity. Scientifically, this work addresses important and longstanding questions about the mechanisms by which the cell microenvironment controls gene expression. Clinically, the long-term impact of this work is to promote the rational development of new biomaterials with mechanical properties tuned for tissue engineering and repair. An additional benefit is the development of an integrated approach using both technologies from engineering and techniques from molecular cell biology for addressing a fundamental question in rigidity sensing. This work is of fundamental interest to diverse fields including cell-biomaterial interactions, nuclear and cell mechanics and molecular and cell biology of gene regulation. The project builds collaboration between the groups of Lele (University of Florida), Nickerson (University of Massachusetts Medical School) and Roux (Sanford Research). Each laboratory brings unique resources to this research including access and expertise in using sophisticated optical and electron microscopes, strong expertise with wet-bench cell and molecular biology experiments specifically related to the nucleus, and expertise in cell and nuclear mechanosensing and biomaterial development and characterization. The team will also benefit from the support of well-known molecular and cell biologists and bioengineers who have pioneered experimental techniques in a broad number of areas in LINC complex biology, nuclear/chromatin structure and function and tissue biomechanics.